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Currently, gene expression analysis can be quite challenging for heterogenous tissues such as tumors.  The tumor microenvironment (TME) is a network of complex interactions between the tumor cells and surrounding immune cells. To identify new predictive biomarkers, it is essential to comprehensively characterize the immune cells within the TME at the molecular level. Traditional methods to assess gene expression in tissues lack either spatial information or sensitivity/specificity.

 

The ACD RNAscope™ in situ hybridization technology combined with the GeoMx® Digital Spatial Profiler (DSP) offers a powerful workflow for highly multiplexed, spatially resolved gene expression analysis. The combined workflow allows users to visualize up to three RNA targets using the RNAscope assay and to identify ‘regions of interest’ (ROI) for further interrogation by GeoMx DSP.

 

Our Joint Workflow Solution

  • The power of single molecule visualization with RNAscope combined with high-plex gene expression analysis using GeoMx DSP RNA assays.
  • Validated protocol with qualified RNAscope probes
  • Molecularly guided high-plex spatial analyses on GeoMx DSP with single cell resolution
  • Integrated manual and automated workflows
  • Fresh frozen and formalin-fixed paraffin-embedded sample compatible

 

 

Technical Overview

  • Visualize mRNA targets such as cytokines, transcription factors, checkpoint markers and immune cell markers using RNAscope for ROI selection
  • Perform GeoMx DSP for targeted genes on selected ROI
  • Quantify expression for 1,800+ gene targets in each region of interest with the GeoMx Cancer Transcriptome Atlas
  • To see a detailed example, refer to the Tech Note ‘Resources’ section below.

 

How to Select Qualified RNAscope Probe Combinations

1.Identify study of interest (e.g. immune checkpoint)

2.Choose from qualified RNAscope probe combinations ( automated or manual ) from the table below or build your own for ROI selection to detect immune cells, tumor cells and soluble factors.

3.Order RNAscope Probes from ACD

 

Qualified RNAscope Probe Combinations Ordering Information

Study of interestChannel 1 TargetChannel 2 TargetChannel 3 TargetFor Automated ProbesFor Manual Probes
Immune checkpointsPDCD1 (PD-1)CD274 (PD-L1)CD3EOrder InformationOrder Information
Tumor vs. StromapanCKPTPRC (CD45)CD3EOrder InformationOrder Information
Cytotxic T Lymphocytes
(Set 1)
CD8AGZMBCD3EOrder InformationOrder Information
Cytotxic T Lymphocytes
(Set 2)
CD8AIFNG-Order InformationOrder Information
T regsCD4FOXP3-Order InformationOrder Information
Activated T regsCD4FOXP3TIGITOrder InformationOrder Information
Immune suppressionTGFB1FOXP3-Order InformationOrder Information
MacrophagesCD68CD163CD3EOrder InformationOrder Information
M1 MacrophagesCD68CXCL10-Order InformationOrder Information
M2 MacrophagesCD163CCL22CD3EOrder InformationOrder Information

 

Resources

Recorded Webinar

Incorporating spatial genomics into your research using the RNAscope technology

Chimeric antigen receptor T (CAR-T) cells are promising treatments for hematologic and solid malignancies. CAR-T cells can recognize and eliminate neoplastic cells expressing specific protein antigens. However, the current generation of CAR-T cells cannot distinguish between neoplastic and normal cells that may also be expressing the same antigen, thus potentially resulting in “on-target/off-tumor” toxicity. In this GEN webinar, we will review the RNAscope® in situ hybridization (ISH) method and show applications of the technology in the CAR-T cell and immuno-oncology fields. We will demonstrate how RNAscope ISH can be utilized to identify novel CAR-T cell targets and subsequently qualify monoclonal antibodies directed against those targets for immunohistochemistry. We will show how ISH can be used to predict CAR-T cell target organ toxicity in preclinical models. Finally, we will discuss optimal preparation of tissues, cell pellets, and cytospin slides for evaluation by ISH. You Will Learn - How RNAscope ISH technology can streamline your target identification and selection workflows - The advantages of using ISH for immunotherapeutic target cell selection - How ISH evaluation of target expression in normal tissues can be used to predict CAR-T cell induced toxicity - Optimal tissue, cell pellet, and cytospin slide preparation for RNAscope ISH

Watch Now

 

Related Literature

White Paper

White Paper "Molecularly Guided Highly Multiplexed Spatial Profiling"

Download pdf

 

Tech Note

Tech Note "Combined Workflow of GeoMx Cancer Transcriptome Atlas and RNAscope Assays"

Download pdf

 

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Advanced Cell Diagnostics

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